1. Field of the Invention
The present invention discloses a reagent kit of global analysis for protein expression and method for qualitative and quantitative proteomic analysis using the same, which combines stable isotope labeling and tandem mass spectrometry to accurately quantify protein expression.
2. Description of Related Art
Protein over-expression is often associated with the presence of disease or the administration of drug. Protein regulation is not only related to the transcribed or translated message, but also the post-translational modification. Currently there are three general approaches to the measurement of protein expression in cells: (1) observing the expression of mRNA by microarray to determine whether the post-transcriptional protein is over-expressed. But this method is based on the premises that mRNA expression is positively correlated with protein expression. However in real biological systems, such positive correlation is rarely present. Thus this method cannot cover all types of protein expression and its reproducibility or accuracy is questionable; (2) carrying out quantitative analysis by using two-dimensional electrophoresis and staining gel separated proteins with coomassie blue stain, silver stain, or immunoblotting. This method is limited by the inherent limitations of the two-dimensional electrophoresis, which shows poor sensitivity and reproducibility in the case of extremely acidic or basic proteins, very large or small proteins, protein expressed at very low level, or membrane proteins; (3) using mass spectrometry in conjunction with chemical reagent (commonly isotope labeling reagent). This method is direct, accurate and reproducible. The commercialized isotope-coded affinity tags (ICAT) reagent is an example.
The technique of culturing cells with isotope which allows isotope to be expressed in proteins through metabolism and thereby observing the protein expression of cells in different environments is widely known. But this method concerns the rate of isotope substitution in cells and is unsuitable for in vivo experiment. The technology of modifying proteins with isotope labeling reagent and then using mass spectrometry for quantitative analysis was conceived in the past five years. As mass spectrometry improves in sensitivity and resolution, it provides a powerful analytical tool as we enter the era of proteomics from genomics. The most noticeable example is the development of ICAT reagent. ICAT comprises an affinity end (e.g. biotin affinity end), which aids in sample purification and binds with the protein reaction group (PRG) through a linker. The linker may be isotopically coded, commonly with hydrogen (1H) and deuterium (2D). This technology based on the different isotopic molecules with the similar chromatographic properties and different mass spectrum is excellent. But the applications of LC/MS are still limited. For examples: (1) the large structure of labeling reagent oftentimes poses difficulty to the fragmentation process in tandem mass spectrometry; (2) the high number of atoms of isotope leads to different retention time in chromatographic column for samples containing hydrogen and deuterium, hence resulting in assay error; and (3) ICAT can only tag cysteine-containing peptides, hence cannot be used in global analysis of proteins not containing cysteine.
Another type of global labeling reagent can bind with peptides of any form, which comprises the steps of derivatizing the N-terminal amino group or C-terminal acid group and then using mass spectrometry for assay. But the majority of assay reagents being reported in literature have the following drawbacks: (1) the mass spectrometric signals of modified peptides are weakened or prohibited; (2) the incomplete labeling reaction or the exchange between isotopes (hydrogen and deuterium) leads to assay error; and (3) the preparation of assay reagent is tedious and the technique has poor compatibility with other separation techniques (e.g. the different retention time of 1H and 2D labeled peptide fragments in liquid chromatographic column affects the quantitative analysis in mass spectrometry).
The assay of phosphoprotein expression comprises the steps of putting specimen in cell medium containing (γ-32P)-ATP and analyzing the change of phosphorylation from the intensity of 32p signals. But this method is time consuming and not entirely safe. A few other technologies that combine stable isotope labeling and mass spectrometry have also been proposed. But their practical applications have room for discussion.